1. Field of the Invention
This invention relates to a system and method for forming skeletal muscle constructs having functional tissue interfaces, such as neuromuscular and myotendinous junctions.
2. Background Art
End-stage organ failure or tissue loss is one of the most devastating and costly problems in medicine. Over 8 million surgical procedures are estimated to be performed each year incurring a health care cost of more than $400 billion annually. Organ and tissue replacement is limited by donor availability and immuno-rejection. Tissue engineering strives to develop biological substitutes that restore, maintain, or improve tissue function. There has been a great deal of activity in the area of in vitro muscle tissue engineering, however, muscle has not been successfully engineered to be functionally or phenotypically equivalent to adult skeletal muscle. Functional data for muscle refers to the active generation of force, work, and power, typically elicited by electrical stimulation. These measures are important in distinguishing muscles of different phenotypes. Developmental muscles are characterized by low excitability and specific force. In adult skeletal muscle, there are two predominant fiber types, slow and fast. Slow muscle has longer contraction and relaxation dynamics than fast muscle and reaches maximal force at a lower frequency of stimulation.
Myosin is the predominant protein in muscle, comprising around 25% of the total protein pool. Myosin is central to muscle function because it is the molecular motor that converts free energy released by the hydrolysis of ATP into mechanical work. Myosin heavy chain (MHC) isoform is the primary determinant of maximum shortening velocity, ATPase activity, maximum power output, and the rate of tension redevelopment. The expression of MHC genes is highly plastic, allowing skeletal muscles to adapt to changing functional needs during development and maturation, exercise training, and repair. A variety of factors, including developmental stage, innervation and the associated neuronal firing patterns, hormonal factors, and mechanical activity/inactivity have been shown to play important regulatory roles in the expression of MHC. During development and maturation, fibers initially express the embryonic MHC isoform, followed by neonatal, and then adult fast or slow isoforms. Chronic low frequency electrical stimulation increases the expression of the adult slow MHC. These data suggest that innervation increases the expression of adult MHC and that loading results in a shift from fast to slow MHC. In fact, it is nearly impossible to change the hormonal environment, innervation, or loading of a muscle in vivo without changing the MHC isoform expression.
In vivo, as the nervous system develops and innervation increases, fetal muscles shift towards the adult phenotype. Just as motor neurons control the expression of MHC in adult muscle, the motor neurons also exert this control during development of muscle cells. The control over fiber type expression is by two distinct mechanisms. The first is a local mechanism involving the release of neural regulatory factors at the level of the neuromuscular junction (NMJ) and the second mechanism is mediated by the activation pattern generated by the nerve. Acetylcholine receptors are among the first proteins expressed during myogenesis. Prior to innervation, acetylcholine receptors are randomly dispersed along the surface of the developing myoblast. During the formation of an NMJ, the chemotrophic, electrical, and mechanical signals between the nerve and muscle induce the aggregation of nicotinic acetylcholine receptors in the plasma membrane of the developing muscle fiber. The maintenance of the NMJ depends on continual cross-talk between the nerve and muscle at the site of nerve-muscle contact.
Tendons are highly organized connective tissues that transmit forces between muscle and bone. They are resilient during the development of tension but flexible enough to conform to their mechanically demanding environment. The mechanical integrity of tendon tissue can be attributed to the parallel fibrils of collagen. In the resting state, the collagen fibrils take on a wavy conformation, defined as the crimp. As a tendon is stretched, the crimped collagen fibrils begin to straighten out, and as a result, the tendon becomes stiffer with increasing application of mechanical strain.
Because of its relatively avascular nature, tendon is a prime candidate for engineered tissue replacement. Previous attempts have been made to create biologically based tendons in vitro, but these have met with limited success because of the difficulty in creating a construct that is both mechanically and biologically compatible with the in vivo environment. Native tendons possess an extracellular matrix (ECM) composed of many proteins, glycosaminoglycans, and proteoglycans that control the assembly of the load-bearing collagen fibril and contribute to the formation of the tissue hierarchy. Fibroblasts rely on cell-matrix signaling pathways during development to properly assemble the fibrils and maintain form and function after maturation.
The myotendinous junction (MTJ) is the structure that transmits force generated by a muscle contraction to the ECM of the muscle and onto the tendon. The morphology of the MTJ is characterized by folding of the sarcolemma into finger like projections at the interface between muscle and tendon at sites of myocyte termination. The projections result in approximately a ten-fold increase in the area of muscle-tendon contact over the cross-sectional area of the muscle fiber and ensure that the stresses experienced by the MTJ are shear stresses thus decreasing the contractile stress applied directly to the sarcolemmal junction. Therefore, the transmission of force between muscle and tendon is rarely associated with a severance at the interface between muscle and tendon, but rather occurs in the body of the muscle, just proximal to the MTJ.
Mechanical transduction of force across the MTJ activates cell signaling pathways that instruct the cells located at the interface to secrete and deposit proteins to form a specialized ECM at the MTJ. The increased expression of several ECM proteins of muscle and tendon, including focal adhesion kinase, paxillin, integrin linked kinase, mitogen-activated protein kinase, and talin, has been shown to occur in response to increased mechanical loading of the MTJ. These proteins provide a conduit by which forces can be transmitted from muscle to tendon. Lack of the expression of these proteins at the MTJ has been shown to lead to structural damage of the interface during contraction.